A functional Rim101 complex is required for proper accumulation of the Ena1 Na+-ATPase protein in response to salt stress in Saccharomyces cerevisiae

[EN] The maintenance of ionic homeostasis is essential for cell viability, thus the activity of plasma membrane ion transporters must be tightly controlled. Previous studies in Saccharomyces cerevisiae revealed that the proper trafficking of several nutrient permeases requires the E3 ubiquitin ligase Rsp5 and, in many cases, the presence of specific adaptor proteins needed for Rsp5 substrate recognition. Among these adaptor proteins are nine members of the arrestin-related trafficking adaptor (ART) family. We studied the possible role of the ART family in the regulation of monovalent cation transporters. We show here that the salt sensitivity phenotype of the rim8/art9 mutant is due to severe defects in Ena1 protein accumulation, which is not attributable to transcriptional defects. Many components of the Rim pathway are required for correct Ena1 accumulation, but not for the accumulation of other nutrient permeases. Moreover, we observe that strains lacking components of the endosomal sorting complexes required for transport (ESCRT) pathway previously described to play a role in Rim complex formation present similar defects in Ena1 accumulation. Our results show that, in response to salt stress, a functional Rim complex via specific ESCRT interactions is required for the proper accumulation of the Ena1 protein, but not induction of the ENA1 gene.This work was supported by grants BFU2011-30197-C03-03 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and EUI2009-04147 [Systems Biology of Microorganisms (SysMo2) European Research Area-Network (ERA-NET)]. V. L-T. was supported by a pre-doctoral fellowship from the Polytechnic University of Valencia.Marqués, MC.; Zamarbide-Forés, S.; Pedelini, L.; Llopis Torregrosa, V.; Yenush, L. (2015). 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Structure—function analysis of the α5 and the α13 helices of human glucokinase: Description of two novel activating mutations

It was recently described that the α5 and the α13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site-directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild-type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the α5 or the α13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme